Cellular lipid homeostasis is required to maintain bilayer fluidity, membrane impermeability, and organelle identity. Disturbances in systemic lipid homeostasis lie at the core of the pathologies for both coronary artery disease and obesity related, type II diabetes. The long-term goal of this research is to translate knowledge of cellular regulatory events to that of the whole organism and to advance our understanding of these increasingly widespread diseases. As a first step toward this goal, we will use cholesterol as a model lipid to understand how cells measure levels of these largely insoluble molecules and then modulate their production. Cholesterol homeostasis in mammalian cells is regulated by a feedback mechanism that monitors the level of cholesterol in membranes and alters transcription of genes required for cholesterol supply. Transcription of these genes is controlled by an ER membrane-bound transcription factor called SREBP that is activated and released from the membrane by proteolysis in sterol-depleted cells. Two additional ER membrane proteins, SCAP and INSIG, act as positive and negative regulators of SREBP activity by controlling ER exit of SREBP and access to Golgi-localized proteases. To date, it is unknown how SCAP and INSIG sense sterols and regulate ER exit of SREBP. To accelerate discovery of novel protein and chemical regulators, we will analyze the function of SREBP, SCAP, and INSIG orthologues in a genetically tractable yeast model in parallel to our studies in mammalian cells. Our sequence database searches reveal that the fission yeast S. pombe, but not the budding yeast S. cerevisiae, contains uncharacterized homologues of SREBP, SCAP, and INSIG: slp1+, scpl+ , and ins1+ , respectively. The primary goal of this proiect is to use S. pombe to isolate unknown regulators of SREBP and SCAP. We hypothesize that Scp1 senses sterols and regulates cleavage of Slp1 through a mechanism similar to that in mammalian cells. In this project, a combination of genetic, molecular, and biochemical approaches will be used to accomplish the following specific aims: 1) To confirm that the S. pombe genes are functional orthologues of mammalian SREBP, SCAP, and INSIG; 2) To define the transcriptional program that Sip1 controls and identify the endogenous chemical regulator of Sip1 activation; 3) To isolate novel genes required for the activation of Sip1; 4) To isolate Scp1 and Ins1 interacting proteins biochemically using affinity purification. The expectation is that novel regulators in S. pombe will have identifiable orthologues in mammals, thus providing new insight into our studies of cholesterol homeostasis in humans and potential therapeutic targets for prevention and treatment of heart disease.